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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Abstract

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.

Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 °C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial "tubes" are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Introduction

Blood flow throughout the brain is regulated by the coordination of vasodilation among cerebral arteries and arterioles in vascular networks1. Endothelial cells lining cerebral resistance arteries command changes in vascular diameter as needed to meet the metabolic demand of neurons1,2,3. In particular, during endothelium-derived hyperpolarization (commonly known as EDH), intracellular Ca2+ ([Ca2+]i) and electrical signaling in endothelial cells coordinate vasodilation among endothelial cells and their surrounding smoot....

Protocol

Before conducting the following experiments, ensure that all animal care use and protocols are approved by the Institutional Animal Care and Use Committee (IACUC) and performed in accord with the National Research Council's "Guide for the Care and Use of Laboratory Animals" (8th Edition, 2011) and the ARRIVE guidelines. The IACUC of Loma Linda University has approved all protocols used for this manuscript for male and female C57BL/6 mice (age range: 3 to 30 mo).

Representative Results

The schematic demonstration of the protocol described above is shown in the attached figures. A brain isolated from a young adult male C57BL/6N mouse (5 months) is shown in Figure 1A. Posterior cerebral arteries are carefully isolated from the Circle of Willis, removed without connective tissue, and cut into segments (Figure 1B-D). From partially digested arterial segments, the intact endothelial tube is produced.......

Discussion

In light of recent developments6,15,16,17, we now demonstrate the method to isolate mouse cerebral arterial endothelium in preparation for simultaneous measurement of [Ca2+]i and Vm underlying EDH consistently for ~2 h at 37 °C. Although technically difficult, we can measure cell-to-cell coupling as well (see reference6, Figure 1). I.......

Acknowledgements

We thank Charles Hewitt for excellent technical assistance while establishing equipment and supplies needed for the current protocols. We thank Drs. Sean M. Wilson and Christopher G. Wilson, from the LLU Center for Perinatal Biology, for providing us with an additional inverted microscope and electrometer, respectively. This research has been supported by National Institutes of Health grant R00-AG047198 (EJB) and Loma Linda University School of Medicine new faculty start-up funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

....

Materials

NameCompanyCatalog NumberComments
GlucoseSigma-Aldrich (St. Louis, MO, USA)G7021
NaClSigmaS7653
MgCl2SigmaM2670
CaCl2Sigma223506
HEPESSigmaH4034
KClSigmaP9541
NaOHSigmaS8045
ATPSigmaA2383
HClThermoFisher Scientific (Pittsburgh, PA, USA)A466250
Collagenase (Type H Blend)SigmaC8051
DithioerythritolSigmaD8255
PapainSigmaP4762
ElastaseSigmaE7885
BSASigmaA7906
Propidium iodideSigmaP4170
DMSOSigmaD8418
Fura-2 AM dyeInvitrogen, Carlsbad, CA, USAF14185
Recirculating chiller (Isotemp 500LCU)ThermoFisher Scientific13874647
Plexiglas superfusion chamber Warner Instruments, Camden, CT, USARC-27
Glass coverslip bottom (2.4 × 5.0 cm)ThermoFisher Scientific12-548-5M
Anodized aluminum platform (diameter: 7.8 cm) Warner InstrumentsPM6 or PH6
Compact aluminum stage Siskiyou, Grants Pass, OR, USA8090P
MicromanipulatorSiskiyou MX10
Stereomicroscopes Zeiss, NY, USAStemi 2000 & 2000-C
Fiber optic light sources Schott, Mainz, Germany & KL200, ZeissFostec 8375
Nikon inverted microscopeNikon Instruments Inc, Melville, NY, USATs2
Phase contrast objectives Nikon Instruments Inc (Ph1 DL; 10X & 20X)
Fluorescent objectives Nikon Instruments Inc20X (S-Fluor), and 40X (Plan Fluor)
Nikon inverted microscopeNikon Instruments IncEclipse TS100
Microsyringe pump controller (Micro4 ) World Precision Instruments (WPI), Sarasota, FL, USASYS-MICRO4
Vibration isolation tableTechnical Manufacturing, Peabody, MA, USA Micro-g
AmplifiersMolecular Devices, Sunnyvale, CA, USAAxoclamp 2B & Axoclamp 900A
Headstages Molecular DevicesHS-2A & HS-9A
Function generator EZ Digital, Seoul, South KoreaFG-8002
Data Acquision SystemMolecular Devices, Sunnyvale, CA, USADigidata 1550A
Audible Baseline MonitorsAmpol US LLC, Sarasota, FL, USA BM-A-TM
Digital Storage OscilloscopeTektronix, Beaverton, Oregon, USA TDS 2024B
Fluorescence System Interface, ARC Lamp + Power Supply, Hyperswitch, PMTMolecular Devices, Sunnyvale, CA, USAIonOptix Systems
Temperature Controller  Warner InstrumentsTC-344B or C
Inline Heater Warner InstrumentsSH- 27B
Valve Controller Warner InstrumentsVC-6
Inline Flow Control ValveWarner Instruments FR-50
Electronic Puller Sutter Instruments, Novato, CA, USAP-97 or P-1000 
MicroforgeNarishige, East Meadow, NY, USA MF-900
Borosilicate Glass Tubes (Trituration)World Precision Instruments (WPI), Sarasota, FL, USA1B100-4
Borosilicate Glass Tubes (Pinning)Warner InstrumentsG150T-6
Borosilicate Glass Tubes (Sharp Electrodes)Warner InstrumentsGC100F-10
Syringe Filter (0.22 µm)  ThermoFisher Scientific722-2520
Glass Petri Dish + Charcoal SylgardLiving Systems Instrumentation, St. Albans City, VT, USADD-90-S-BLK
Vannas Style Scissors (3 mm & 9.5 mm)World Precision Instruments555640S, 14364
Scissors 3 & 7 mm bladesFine Science Tools (or FST), Foster City, CA, USAMoria MC52 & 15000-00
Sharpened fine-tipped forceps FSTDumont #5 & Dumont #55

References

  1. Longden, T. A., Hill-Eubanks, D. C., Nelson, M. T. Ion channel networks in the control of cerebral blood flow. Journal of Cerebral Blood Flow & Metabolism. 36 (3), 492-512 (2016).
  2. Chen, B. R., Kozberg, M. G., Bouchard, M. B., Shaik, M. A., Hillman, E. M.

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